The phosphoinositide 3-kinase (PI3K)-AKT pathway is activated in human cancers, commonly through genetic alterations of its many signalling components. The p110α catalytic subunit of PI3K is mutated or amplified and overexpressed in ovarian, breast, cervical, brain and colorectal cancers (Campbell et al., 2004; Samuels et al., 2004). AKT is amplified and overexpressed in breast, ovarian and pancreatic cancer (Bellacosa et al., 1995; Cheng et al., 1992; Miwa et al., 1996; Ruggeri et al., 1998). PTEN, the key PI3K-AKT pathway antagonist, is frequently targeted for somatic inactivation in a broad spectrum of human cancers (Cully et al., 2006). Moreover, germline PTEN mutations define three related syndromes (Cowden disease, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome) characterized by hamartomatous growths of multiple cell types, increased cancer incidence and developmental defects (Wanner et al., 2001). PTEN's diverse tumor suppressor role in different cell lineages is further evidenced by the cancer-prone condition in germline and conditional Pten mutant mice, which develop a broad range of tumors including carcinomas of the skin, prostate, colon, breast, and endometrium, as well as thymic lymphomas, among other cancers (Di Cristofano et al., 1998; Li et al., 2002; Podsypanina et al., 1999; Suzuki et al., 2001; Wang et al., 2003; You et al., 2002).
In addition to a direct role in tumor development, the PI3K-AKT signalling network has been linked to endothelial cell homeostasis (Shiojima and Walsh, 2002). At least one of the three AKT homologues is highly expressed in endothelial cells and stimulates eNOS and survivin expression to promote survival (Dimmeler et al., 1999; Fulton et al., 1999; Tran et al., 2002). Consistent with these observations, one of the PTEN germline mutation syndromes, Bannayan-Zonana, is characterized by hemangiomas (hamartomas of the endothelial cell lineage) in diverse tissues including the deep viscera (Wanner et al., 2001).
The wide range of benign to malignant phenotypes mediated by PI3K-PTEN-AKT signalling is consistent with the existence of diverse downstream effectors that are likely to be differentially utilized in conferring neoplastic phenotypes in distinct cell lineages. Among such potential effectors are AKT phosphorylation targets TSC2, GSK3 and the FoxOs (Brunet et al., 1999; Cross et al., 1995; Inoki et al., 2002). Recently, much attention has been focused on TSC2 and the mTOR signalling axis as mounting pharmacological evidence suggests that this axis may be the prime effector of the PI3K-AKT pathway, with TSC2 serving as a central node linking LKB1-AMPK and PI3K-AKT with mTOR (Hay, 2005). Indeed, the potent anti-neoplastic impact of pharmacologic mTOR inhibition has raised questions as to the relevance of other downstream AKT targets, particularly the FoxOs, in the development of cancer. On the other hand, the FoxO effector arm has been shown to control cell number in Drosophila (Junger et al., 2003; Puig et al., 2003), and recent studies have implicated the FoxO transcription factors in mediating some of the growth and survival-promoting effects of AKT signaling in endothelial cells (Potente et al., 2005; Skurk et al., 2004). Therefore, the relevance of FoxO transcription factors in cancer and their roles in normal tissue homeostasis remain to be elucidated.
Whereas C. elegans and D. melanogaster contain a single FoxO gene (DAF-16 and dFOXO, respectively), mice and humans possess three highly related FoxO homologues (FoxO1, FoxO3, and FoxO4) with overlapping patterns of expression and transcriptional activities (Anderson et al., 1998; Biggs et al., 2001; Furuyama et al., 2000). A fourth more distantly related mammalian FoxO family member, FoxO6, has been identified, although it appears to be regulated by distinct mechanisms and its expression is more highly restricted to the brain (Jacobs et al., 2003; van der Heide et al., 2005). Activation of PI3K by extracellular growth signals leads to FoxO phosphorylation by Akt, whereupon the FoxOs are translocated from the nucleus to the cytoplasm and thereby inactivated.
In the nucleus, the FoxOs regulate cell survival and cell cycle progression through direct positive and negative transcriptional control of specific gene targets that are wired into diverse cancer regulatory pathways. The FoxOs promote apoptosis via up-regulation of FasL (Alvarez et al., 2001; Brunet et al., 1999; Siegel et al., 2000) and Bim (Dijkers et al., 2002; Dijkers et al., 2000a; Stahl et al., 2002), as well as down-regulation of the pro-survival factor BCL-xL (Tang et al., 2002). In cell cycle regulation, enforced FoxO expression results in G1 arrest through increased expression of the cyclin-dependent kinase inhibitor p27kip1 and down-regulation of D-type cyclins (Medema et al., 2000) (Schmidt et al., 2002). The FoxOs have also been linked to other cancer-relevant pathways, such as the NFκB and TGF-β pathways. The IκB kinase phosphorylates and inhibits the FoxO factors, rationalizing some of the growth-promoting properties of IκB kinase (Hu et al., 2004). The FoxO factors also associate with Smad proteins, which are activated by TGF-β signaling (Seoane et al., 2004), to enhance transcription of the cell cycle inhibitor, p21Cip1.
The FoxO1, FoxO3, and FoxO4 proteins behave similarly in biochemical studies, regulate common target genes, and bind to the same target DNA sequence (Biggs et al., 2001; Brunet et al., 1999; Furuyama et al., 2000). At the same time, mouse FoxO knockout mutants have revealed unique roles for the FoxOs, such as the requirement for FoxO3 in the regulation of ovarian primordial follicle activation (Castrillon et al., 2003; Hosaka et al., 2004) and for FoxO1 in vascular development during embryogenesis (Furuyama et al., 2004; Potente et al. 2005). However, while the three FoxOs serve some discrete functions, they are also likely to have significant functional redundancies, particularly as they are broadly expressed during embryonic development and in adult tissues (Furuyama et al., 2000). In this regard, conventional genetic analysis can fail to uncover important biological functions among closely-related gene families, as has been demonstrated for the Rb/p107/p130 (Lee et al., 1996; Sage et al., 2000) and p53/p63/p73 gene families (Flores et al., 2005; Flores et al., 2002). Indeed, many analogies can be drawn between the FoxO and the Rb, and p53 gene families, all of which regulate cell survival and growth pathways relevant to cancer and consist of members with common physiological roles.
In the context of the above biochemical and biological data linking the FoxOs to key cancer signaling pathways, initial indications that various FoxO knockout mice do not show an overt tumor-prone phenotype were somewhat unanticipated (Castrillon et al., 2003; Furukawa-Hibi et al., 2002; Hosaka et al., 2004; Lin et al., 2004). While this may relate to the physical and functional relatedness and overlapping patterns of expression of the FoxO members, it is formally possible that the FoxO arm of the PI3K-AKT signaling network plays a relatively minor role in cancer suppression and vascular biology relative to other AKT downstream targets. To address these issues, we have generated conditional alleles for all three FoxO members with which to conduct a systematic evaluation of FoxO family function in vivo.